The objective of the proposed research is to analyze the reaction mechanism of enzymatic pyrophosphoryl transfers. Transfer of an intact pyrophosphoryl group from ATP occurs in the biosynthesis of the central metabolite PRPP and in the biosynthesis of the coenzyme forms of two vitamins, folic acid and thiamin. Research will concentrate on determining if covalent pyrophosphoryl-enzyme (E-PP) complexes are formed as intermediates during catalysis. Direct isolation of E-PP complexes will be attempted, primarily by gel filtration with doubly- labeled substrates. Any reactive intermediates will be tested for their chemical and kinetic competence in transfer of the bound PP group to either substrate in the given enzymatic reaction. E-PP species will be examined for their chemical linkages and for the enzymic residues utilized in formation of these energetic intermediates. In addition to the three overt pyrophosphokinases alluded to above, two other enzymes, which may be crypto-pyrophosphotransferases, will be studied: namely,PEP synthase and pyruvate phosphate dikinase. The significance of the proposed research, given that covalent catalysis is operant, would be an understanding of the generalized mode of catalysis in reactions where group transfer potential is transferred to metabolites from ATP in which nucleophiles attack at the beta phosphorous of ATP rather than at the alpha (adenylyl transfer) or at the gamma phosphate (phosphoryl transfer). This will further our knowledge of how organisms use the "high energy" anhydride linkages in ATP to drive the biosynthesis of two coenzymes and, via PRPP, such crucial metabolites as purines and pyrimidines.